The aggressiveness of succinate dehydrogenase subunit B-deficient chromaffin cells is reduced when their bioelectrical properties are restored by glibenclamide.

Pheochromocytomas/paragangliomas (PPGLs) are neuroendocrine tumours, mostly resulting from mutations in predisposing genes. Mutations of succinate dehydrogenase (SDH) subunit B (SDHB) are associated with high probability of metastatic disease. Since bioelectrical properties and signalling in cancer are an emerging field, we investigated the metabolic, functional and electrophysiological characteristics in human succinate dehydrogenase subunit B (SDHB)-deficient pheochromocytoma cells. These cells exhibited reduced SDH function with elevated succinate-to-fumarate ratio and reduced intracellular ATP levels. The analysis of membrane passive properties revealed a more hyperpolarized membrane potential and a lower cell capacitance of SDHB-deficient cells compared to the parental ones. These bioelectrical changes were associated with reduced proliferation and adhesion capacity of SDHB-deficient cells. Only in SDHB-deficient cells, we also observed an increased amplitude of potassium currents suggesting an activation of ATP-sensitive potassium channels (KATP). Indeed, exposure of the SDHB-deficient cells to glibenclamide, a specific KATP inhibitor, or to ATP caused normalization of potassium current features and altered proliferation and adhesion. In this work, we show for the first time that reduced intracellular ATP levels in SDHB-deficient chromaffin cells impaired cell bioelectrical properties, which, in turn, are associated with an increased cell aggressiveness. Moreover, we first ever demonstrated that glibenclamide not only reduced the outward potassium currents in SDHB-deficient cells but increased their growth capacity, reduced their ability to migrate and shifted their phenotype towards one more similar to that of parental one.

Hemodynamic parameters in patients undergoing surgery for pheochromocytoma/paraganglioma: a retrospective study.

BACKGROUND: Pheochromocytoma (PHEO) and paraganglioma (PGL) are rare neuroendocrine tumors characterized by hemodynamic instability, caused by the paroxysmal release of catecholamines. Patients may develop cardiovascular complications in the perioperative phase due to the massive release of catecholamines, particularly during anesthetic induction and surgical manipulation of the tumor. The aim of this retrospective study was to evaluate the risk factors involved in perioperative hemodynamic instability in patients who underwent surgery for chromaffin tumors. METHODS: Forty patients (median age 55 [36.50-64.50]) undergone surgery for PHEO/abdominal PGL from January 2011 to December 2016 at the AOU Careggi (Florence, Italy) were retrospectively evaluated. Systolic, diastolic, and mean blood pressure were considered at baseline and during surgery. Patients with blood pressure steadily < 140/90 mmHg before surgery were considered "adequately prepared". A preoperative therapy with doxazosin, a selective alpha-1 blocker, was started in all patients for at least 14 days prior to the surgery. The presence of hemodynamic instability was reported. RESULTS: Comparing males and females, a significant difference in doxazosin daily dose (p = 0.018), systolic blood pressure (p = 0.048), and in the proportion of adequately prepared patients (p = 0.031) emerged. A positive correlation between preoperative daily dose of doxazosin, tumor size (B = 0.60, p < 0.001), and urinary normetanephrine levels (B = 0.64, p < 0.001) was also observed. Hemodynamic instability occurred in 30.0% of patients. The absence of adequate preparation (p = 0.012) before surgery, urinary normetanephrine levels (NMNur p = 0.039), and surgery time (minutes) (p = 0.021) resulted as risk factors of hemodynamic instability in our series. The use of intraoperative drugs was higher in patients with hemodynamic instability (p < 0.001). A pre-surgical SBP level of > 133 mmHg (OR = 6 CI95% 1.37-26.20, p = 0.017) and an intraoperative SBP and MBP levels of > 127 mmHg (OR = 28.80 CI95% 2.23-371.0, p = 0.010) and > 90 mmHg (OR = 18.90 CI95% 1.82-196.0, p = 0.014), respectively, were identified as effective thresholds to recognize patients at higher risk of HI. CONCLUSIONS: A preoperative therapy with alpha-blockers is useful, but not sufficient to avoid surgical risks. Patients with higher pre-surgical levels of NMNur, pre-surgical SBP > 133 mmHg, and/or intraoperative SBP > 127 mmHg and MBP > 90 mmHg, should be carefully monitored. A multidisciplinary approach is indispensable to optimize the management of PHEOs/abdominal PGLs in order to reduce surgical complications.

Tumour microenvironment in pheochromocytoma and paraganglioma.

Pheochromocytomas and Paragangliomas (Pheo/PGL) are rare catecholamine-producing tumours derived from adrenal medulla or from the extra-adrenal paraganglia respectively. Around 10-15% of Pheo/PGL develop metastatic forms and have a poor prognosis with a 37% of mortality rate at 5 years. These tumours have a strong genetic determinism, and the presence of succinate dehydrogenase B (SDHB) mutations are highly associated with metastatic forms. To date, no effective treatment is present for metastatic forms. In addition to cancer cells, the tumour microenvironment (TME) is also composed of non-neoplastic cells and non-cellular components, which are essential for tumour initiation and progression in multiple cancers, including Pheo/PGL. This review, for the first time, provides an overview of the roles of TME cells such as cancer-associated fibroblasts (CAFs) and tumour-associated macrophages (TAMs) on Pheo/PGL growth and progression. Moreover, the functions of the non-cellular components of the TME, among which the most representatives are growth factors, extracellular vesicles and extracellular matrix (ECM) are explored. The importance of succinate as an oncometabolite is emerging and since Pheo/PGL SDH mutated accumulate high levels of succinate, the role of succinate and of its receptor (SUCNR1) in the modulation of the carcinogenesis process is also analysed. Further understanding of the mechanism behind the complicated effects of TME on Pheo/PGL growth and spread could suggest novel therapeutic targets for further clinical treatments.

The regulatory effect of sex steroids on the RhoA/ROCK pathway in the rat distal vagina.

BACKGROUND: Sex steroids have been demonstrated as important modulators of vaginal function. The RhoA/ROCK calcium-sensitizing pathway plays a role in genital smooth muscle contractile mechanism, but its regulation has never been elucidated. AIM: This study investigated the sex steroid regulation of the vaginal smooth muscle RhoA/ROCK pathway using a validated animal model. METHODS: Ovariectomized (OVX) Sprague-Dawley rats were treated with 17ß-estradiol (E2), testosterone (T), and T with letrozole (T + L) and compared with intact animals. Contractility studies were performed to test the effect of the ROCK inhibitor Y-27632 and the nitric oxide (NO) synthase inhibitor L-NAME. In vaginal tissues, ROCK1 immunolocalization was investigated; mRNA expression was analyzed by semiquantitative reverse transcriptase-polymerase chain reaction; and RhoA membrane translocation was evaluated by Western blot. Finally, rat vaginal smooth muscle cells (rvSMCs) were isolated from the distal vagina of intact and OVX animals, and quantification of the RhoA inhibitory protein RhoGDI was performed after stimulation with NO donor sodium nitroprusside, with or without administration of the soluble guanylate cyclase inhibitor ODQ or PRKG1 inhibitor KT5823. OUTCOMES: Androgens are critical in inhibiting the RhoA/ROCK pathway of the smooth muscle compartment in the distal vagina. RESULTS: ROCK1 was immunolocalized in the smooth muscle bundles and blood vessel wall of the vagina, with weak positivity detected in the epithelium. Y-27632 induced a dose-dependent relaxation of noradrenaline precontracted vaginal strips, decreased by OVX and restored by E2, while T and T + L decreased it below the OVX level. In Western blot analysis, when compared with control, OVX significantly induced RhoA activation, as revealed by its membrane translocation, with T reverting it at a level significantly lower than in controls. This effect was not exerted by E2. Abolishing NO formation via L-NAME increased Y-27632 responsiveness in the OVX + T group; L-NAME had partial effects in controls while not modulating Y-27632 responsiveness in the OVX and OVX + E2 groups. Finally, stimulation of rvSMCs from control animals with sodium nitroprusside significantly increased RhoGDI protein expression, counteracted by ODQ and partially by KT5823 incubation; no effect was observed in rvSMCs from OVX rats. CLINICAL IMPLICATIONS: Androgens, by inhibiting the RhoA/ROCK pathway, could positively contribute to vaginal smooth muscle relaxation, favoring sexual intercourse. STRENGTHS AND LIMITATIONS: This study describes the role of androgens in maintaining vaginal well-being. The absence of a sham-operated animal group and the use of the only intact animal as control represented a limitation to the study.

Metformin Treatment Induces Different Response in Pheochromocytoma/Paraganglioma Tumour Cells and in Primary Fibroblasts.

Pheochromocytoma/paragangliomas (PPGLs) are neuroendocrine tumours, often non-metastatic, but without available effective treatment for their metastatic form. Recent studies have shown that metformin exhibits antiproliferative activity in many human cancers, including PPGLs. Nevertheless, no data are available on the role of metformin on PPGL cells (two-dimension, 2D) and spheroids (three-dimension, 3D) migration/invasion. In this study, we observed that metformin exerts an antiproliferative effect on 2D and 3D cultures of pheochromocytoma mouse tumour tissue (MTT), either silenced or not for the SDHB subunit. However, metformin did not affect MTT migration. On the other hand, metformin did not have a short-term effect on the proliferation of mouse primary fibroblasts, but significantly decreased their ability to migrate. Although the metabolic changes induced by metformin were similar between MTT and fibroblasts (i.e., an overall decrease of ATP production and an increase in intracellular lactate concentration) the activated signalling pathways were different. Indeed, after metformin administration, MTT showed a reduced phosphorylation of Akt and Erk1/2, while fibroblasts exhibited a downregulation of N-Cadherin and an upregulation of E-Cadherin. Herein, we demonstrated that metformin has different effects on cell growth and spread depending on the cell type nature, underlining the importance of the tumour microenvironment in dictating the drug response.

SDHB and SDHD silenced pheochromocytoma spheroids respond differently to tumour microenvironment and their aggressiveness is inhibited by impairing stroma metabolism.

Germline mutations in more than 20 genes, including those encoding for the succinate dehydrogenase (SDH), predispose to rare tumours, such as pheochromocytoma/paraganglioma (PPGL). Despite encoding for the same enzymatic complex, SDHC and SDHD mutated PHEO/PGLs are generally benign, while up to 80% of SDHB mutated ones are malignant. In this study, we evaluated the different effects of tumour microenvironment on tumour cell migration/invasion, by co-culturing SDHB or SDHD silenced tumour spheroids with primary cancer-associated fibroblasts (CAFs). We observed that SDHD silenced spheroids had an intermediate migration pattern, compared to the highest migration capability of SDHB and the lowest one of the wild type (Wt) spheroids. Interestingly, we noticed that co-culturing Wt, SDHB and SDHD silenced spheroids with CAFs in low glucose (1 g/l) medium, caused a decreased migration of all the spheroids, but only for SDHB silenced ones this reduction was significant. Moreover, the collective migration, observed in high glucose (4.5 g/l) and characteristic of the SDHB silenced cells, was completely lost in low glucose. Importantly, migration could not be recovered even adding glucose (3.5 g/l) to low glucose conditioned medium. When we investigated cell metabolism, we found that low glucose concentration led to a reduction of oxygen consumption rate (OCR), basal and maximal oxidative metabolism, and ATP production only in CAFs, but not in tumour cells. These results suggest that CAFs metabolism impairment was responsible for the decreased invasion process of tumour cells, most likely preventing the release of the pro-migratory factors produced by CAFs. In conclusion, the interplay between CAFs and tumour cells is distinctive depending on the gene involved, and highlights the possibility to inhibit CAF-induced migration by impairing CAFs metabolism, indicating new potential therapeutic scenarios for medical therapy.

A rare case of Krukenberg tumor by gallbladder cancer.

INTRODUCTION: Gallbladder cancer commonly spreads by direct extension to the liver and adjacent organs of the gastrointestinal tract. Ovarian metastases by biliary origin, though known, are a very uncommon finding. PRESENTATION OF CASE: We report a rare metastatic localization by gallbladder cancer, Krukenberg tumor mimicking a primitive ovarian cancer. A comprehensive critical review was performed and suggested strategies were analyzed. DISCUSSION: The prognosis of ovarian metastastes by biliary origin is very poor with an overall survival estimated at around 6 months. The variable clinical presentation, radiology and serum markers make the appropriate histological diagnosis mandatory. CONCLUSION: The presence of Krukenberg tumor should be considered in the work-up of gallbladder cancer.

Complex multisystem phenotype associated with the mitochondrial DNA m.5522G>A mutation.

Mitochondrial tRNAs are responsible for more than half of pathogenic point mutations in the mitochondrial genome (mtDNA). Different mutations give rise to widely differing phenotypes, ranging from isolated organ-specific diseases to multisystem conditions. Herein, we report a 40-year-old woman presenting with a complex multisystem phenotype including sensorineural hearing loss, retinopathy, severe dilated cardiomyopathy, non-insulin dependent diabetes mellitus, and renal failure. Sequence analysis of mtDNA identified the m.5522G>A mutation in MT-TW, the gene encoding mitochondrial tRNA for tryptophan. The heteroplasmic variant, thus far described once, was almost exclusively confined to skeletal muscle tissue, as shown by massive parallel sequencing and corroborated by an ad hoc designed PCR-based strategy. This patient, presenting a severe, multisystem involvement apparently sparing the brain, contributes to the genetic heterogeneity of mitochondrial diseases caused by mutations in mitochondrial tRNAs.

Effects of ultralow topical estriol dose on vaginal health and quality of life in postmenopausal women who underwent surgical treatment for pelvic organ prolapse.

OBJECTIVE: To evaluate the efficacy of low concentrations of vaginal estriol gel in postmenopausal women with pelvic static disorders before and after vaginal surgical treatment, assessing vaginal health, sexual function, and quality of life (QoL). METHODS: Women affected by genital prolapse were enrolled. Vaginal health, QoL, and sexual function were investigated at baseline (T0), before surgery (T1), and 13 weeks after surgery (T2). At baseline, participants were randomized 1:1. Women in group A (38 women) were treated daily with vaginal gel containing 50 µg estriol for 12 weeks and women in group B (37 women) did not receive any estrogen treatment. After this period and before surgery, a first examination was carried out (T1). One week after surgical treatment, group A underwent randomization 1:1 to group A1 repeating estriol vaginal gel for 12 weeks, and group A2 discontinuing the estrogen treatment. The second follow-up examination (T2) was performed at the 13th week after surgery. RESULTS: All aspects of vaginal health improved in group A on estriol before surgery with respect to baseline (P < 0.001). After surgery, 17 participants of group A1, 16 of group A2, and 30 of group B completed the study. Group A1 (on estriol plus surgery) further improved with respect to the presurgery estriol treatment (P < 0.01). Moreover, group A2 (T2) experienced a worsening of vaginal health versus intragroup presurgery estriol treatment (P < 0.01), and versus intergroup surgical estriol treatment (P < 0.05). QoL improved in women only after surgery, with (P < 0.01) or without (P < 0.05) estriol treatment. Finally, the sexual function of participants on estriol before surgery did not change. On the contrary, it improved after surgery in both participants on estriol (P < 0.001) and without estriol (P < 0.01). Moreover, surgical estriol participants had a better score than surgical no-estriol participants (P < 0.05). CONCLUSIONS: Estriol vaginal gel (0.005%) administration significantly improved the vaginal health of natural postmenopausal women before and after vaginal surgery. Both sexual health and QoL also significantly improved after surgery.

Validation of the Minority Stress Scale Among Italian Gay and Bisexual Men.

The experience of sexual orientation stigma (e.g., homophobic discrimination and physical aggression) generates minority stress, a chronic form of psychosocial stress. Minority stress has been shown to have a negative effect on gay and bisexual men's (GBM's) mental and physical health, increasing the rates of depression, suicidal ideation, and HIV risk behaviors. In conservative religious settings, such as Italy, sexual orientation stigma can be more frequently and/or more intensively experienced. However, minority stress among Italian GBM remains understudied. The aim of this study was to explore the dimensionality, internal reliability, and convergent validity of the Minority Stress Scale (MSS), a comprehensive instrument designed to assess the manifestations of sexual orientation stigma. The MSS consists of 50 items assessing (a) Structural Stigma, (b) Enacted Stigma, (c) Expectations of Discrimination, (d) Sexual Orientation Concealment, (e) Internalized Homophobia Toward Others, (f) Internalized Homophobia toward Oneself, and (g) Stigma Awareness. We recruited an online sample of 451 Italian GBM to take the MSS. We tested convergent validity using the Perceived Stress Questionnaire. Through exploratory factor analysis, we extracted the 7 theoretical factors and an additional 3-item factor assessing Expectations of Discrimination From Family Members. The MSS factors showed good internal reliability (ordinal α > .81) and good convergent validity. Our scale can be suitable for applications in research settings, psychosocial interventions, and, potentially, in clinical practice. Future studies will be conducted to further investigate the properties of the MSS, exploring the association with additional health-related measures (e.g., depressive symptoms and anxiety).

Rare cause of odynophagia: Giant esophageal ulcer.

Gastrointestinal complications are a frequent cause of morbidity after transplantation and may affect up to 40% of kidney transplant recipients. Here we report a rare case of idiopathic giant esophageal ulcer in a kidney transplant recipient. A 37-year-old female presented with a one-week history of odynophagia and weight loss. Upon admission, the patient presented cold sores, and a quantitative cytomegalovirus polymerase chain reaction was positive (10(5) copies/mL). An upper endoscopy demonstrated the presence of a giant ulcer. Serological test and tissue biopsies were unable to demonstrate an infectious origin of the ulcer. Immunosuppression was reduced and everolimus was introduced. An empirical i.v. therapy with acyclovir was started, resulting in a dramatic improvement in symptoms and complete healing of the ulcer. Only two cases of idiopathic giant esophageal ulcer in kidney transplant recipients have been reported in the literature; in both cases, steroid therapy was successful without recurrence of symptoms or endoscopic findings. However, this report suggests that correction of immune imbalance is mandatory to treat such a rare complication.

Quality of life and sexual function of naturally postmenopausal women on an ultralow-concentration estriol vaginal gel.

OBJECTIVE: This study aims to evaluate the sexual function and quality of life (QoL) of naturally postmenopausal women affected by genitourinary syndrome of menopause who were treated with an ultralow-concentration estriol vaginal gel (0.005%). METHODS: Postmenopausal women with vulvovaginal atrophy symptoms and sexual disorders were enrolled in a case-control study. Women were treated with vaginal gel (containing 50 µg of estriol) daily for 3 weeks and then twice weekly up to 12 weeks. Determination of vaginal maturation index, evaluation of vaginal pH, and assessment of vaginal atrophy symptoms were carried out. QoL, sexual function, and distress were investigated using the Short Form 36, Female Sexual Function Index, and Female Sexual Distress Scale questionnaires. Changes between baseline and week 12 were assessed. RESULTS: Sixty-eight women were included in the study group, and 42 women were included in the control group. Women on estriol vaginal gel had a significant increase in vaginal maturation index and improvement of vaginal pH compared with baseline (P < 0.05). Mean total Female Sexual Function Index score improved, and Female Sexual Distress Scale score decreased from baseline to follow-up. Results from the Short Form 36 questionnaire showed a significant improvement in the overall index of somatic aspects (P < 0.05). The control group showed no changes from baseline evaluation (P = NS). CONCLUSIONS: Estriol vaginal gel (0.005%) therapy significantly improves the trophism of the vaginal mucosa and the sexual health and QoL of naturally postmenopausal women. These results confirm that low doses of vaginal estrogen must be considered as the first choice for the initial treatment of postmenopausal genitourinary symptoms.

Visual scanning behavior is related to recognition performance for own- and other-age faces.

It is well-established that our recognition ability is enhanced for faces belonging to familiar categories, such as own-race faces and own-age faces. Recent evidence suggests that, for race, the recognition bias is also accompanied by different visual scanning strategies for own- compared to other-race faces. Here, we tested the hypothesis that these differences in visual scanning patterns extend also to the comparison between own and other-age faces and contribute to the own-age recognition advantage. Participants (young adults with limited experience with infants) were tested in an old/new recognition memory task where they encoded and subsequently recognized a series of adult and infant faces while their eye movements were recorded. Consistent with findings on the other-race bias, we found evidence of an own-age bias in recognition which was accompanied by differential scanning patterns, and consequently differential encoding strategies, for own-compared to other-age faces. Gaze patterns for own-age faces involved a more dynamic sampling of the internal features and longer viewing time on the eye region compared to the other regions of the face. This latter strategy was extensively employed during learning (vs. recognition) and was positively correlated to discriminability. These results suggest that deeply encoding the eye region is functional for recognition and that the own-age bias is evident not only in differential recognition performance, but also in the employment of different sampling strategies found to be effective for accurate recognition.